Mental retardation with macroorchidism and Xq28 fragile site is a common X-linked disease that causes retardation in one per 2000 males. Affected individuals demonstrate a characteristic non-staining gap or "fragile site" at band Xq28 of metaphase chromosome preparations. This cytogenetic abnormality is an inducible phenomenon that depends on appropriate culture conditions including limited folate or exposure to methotrexate or fluorodeoxyuridine for expression. The site is seen in 50% or less of metaphase spreads from an affected individual and significant variation exists in the fraction of cells demonstrating the site in different cell types and between affected male siblings. Heterozygote females may be retarded and express the site, although at a lower rate than males, or not express at all. Using somatic cell hybridization techniques, we have shown that Chinese hamster V79 cells containing only a human X chromosome from an affected male will demonstrate the site. We propose to use the somatic cell hybrid system to: 1) Develop optimum expression conditions in defined media with dialyzed fetal calf serum to take advantage of the hybrids' rapid, non-fastidious growth. 2) Analyze the role of cell cycle by synchronizing the cells without using DNA synthesis inhibitors which might obscure the results. 3) Study X chromosomes from affected males and females in different families and from within a family in a uniform xenogenic background to determine whether there are human autosomal or other X-linked loci that modify expression rates. 4) Determine the effect of X-inactivation on fragile site expression in obligate heterozygotes by studying active and inactive X chromosomes singly or together in rodent human hybrids. 5) Characterize the mechanism of induction of the site by placing affected X chromosomes in rodent cells with defined mutations in purine and pyrimidine metabolism. 6) Use nucleases as probes of the chromatin structure at the fragile site with the goal of identifying, isolating and characterizing the DNA from the fragile site. 7) Examine the ultrastructure of the fragile site, specifically, the arrangement of the 30 nm chromatin fiber within the lesion using high-resolution scanning electron microscopy.